Alcoholic liver damage (ALD) is among the top ten causes of death in the USA, but existing therapeutic interventions are relatively ineffective. Hepatocytes are susceptible to damage probably because they have a high level of alcohol dehydrogenase (ADH) which metabolizes alcohol to form acetaldehyde and reactive oxygen species (ROS), which are both cytotoxic. Close similarities between eukaryotic cells suggest that alcohol-resistant plant cells are a potential source of hepatoprotective compounds, particularly if these plant cells have been "engineered" to metabolize alcohol rapidly by ADH. In the preceding phase 1 research, transgenic tobacco cells expressing rat liver ADH, and a cellular screen for hepatoprotection were developed. In this phase 2 proposal, mutant cultures from this transgenic plant will be selected for resistance to alcohol-induced toxicity. Extracts from this resistant sub-population will then be screened for the presence of cytoprotective metabolites against alcohol-induced toxicity in hepatocytes. Greater genetic diversity can be introduced by investigation of protective metabolites that have been evolved "naturally" by plants. In aquatic plant species, fermentation reactions become important energy sources, particularly in root tissue, when oxygen tension is low. This induces ADH, with prolonged very high rates of alcohol metabolism to acetaldehyde (Vartapetian &Jackson, 1997). Some of these species should have evolved metabolites that protect against intracellular generation of ROS and acetaldehyde, and some of these might also protect hepatocytes from alcohol. Extracts from ~75 native aquatic plant species were immediately available to the project, and ~30 have now been screened. Several of these extracts are highly protective to hepatocytes. In phase 2 this screening will continue, and the two approaches will be combined by culturing and selecting mutant populations of those aquatic plant species with greatest hepatoprotective activity. Subsequently, the protective activity in these transgenic plants will be optimized, and extracts and semi-purified compounds evaluated in animal models of ALD in vivo. Several patentable therapeutic products are predicted, which will be commercialized with a partner in phase 3. The development of the technology also extends existing intellectual property licensed exclusively to Naprogenix by the University of Kentucky Research Foundation.